Enhancer of anti-tumor effect

ABSTRACT

A nucleotide selected from the group consisting of 3&#39;-deoxyguanosine-5&#39;-monophosphate, 3&#39;-deoxyadenosine-5&#39;-monophosphate and pharmaceutically-acceptable salts thereof enhances, when administered to a tumor-bearing animal under an anti-tumor treatment, the anti-tumor effect due to the anti-tumor treatment. The anti-tumor treatment comprises irradiation of a tumor site of the animal or administration of an anti-tumor agent to the animal.

BACKGROUND OF THE INVENTION

1. Field of the Art

This invention relates to an enhancer of anti-tumor effect.

In the art of treatment of tumors, there have been many developmentsfrom various aspects. In radiotherapy, as a branch of the development,there have also been attempts to improve the results of therapy. As onemethod, it is proposed to improve geometrical distribution by use ofsuch methods as radiation of accelerated heavy ion particles or πmesons. Another approach now under the development is to enhanceselectively the sensitivity of tumor cells under hypoxic conditions,which are most resistant to radiotherapy among tumors, by utilizing ahypoxic cell sensitizer. Alternatively, combination treatmentsincorporating a method utilizing other anti-tumor factors, such ashyperthermia or chemotherapy have been attempted.

However, in the method for improvement of geometrical dose distribution,it is required to use enormous funds for installation of an acceleratorand auxiliary equipment necessary for practicing the method as well as alarge personnel requirements, including expert engineers and physicians.Other methods also involve drawbacks such as great damage to normalcells. For example, misonidazole, which is a hypoxic cell sensitizer,has neurotoxicity, and hence it is difficult to administer it in a largequantity, whereby no great radiosensitizing effect can be expected atconcentrations available in clinical use, its effect being small in alow dose range (less than 1000 rad) as employed in a routine therapy.

In view of the fact that the tumor cells with radioresistance underhypoxic conditions are at a quiescent stage, I have investigated thepossibility of radiosensitization by inhibiting potentially lethaldamage repair (hereinafter referred to as "PLDR"), which is especiallymarked in quiescent cells. During the course of this investigation,3'-deoxyadenosine (cordycepin, herein referred to as "3'-dAdo") wasconfirmed to have a PLDR-inhibiting ability. However, 3'-dAdo is readilyinactivated by adenosinedeaminase in bodies. I found that thePLDR-inhibiting ability of 3'-dAdo can be strengthened and prolonged byusing it in combination with 2'-deoxycoformycin, which is an inhibitoragainst adenosinedeaminase. However, there is also a report that2'-deoxycoformycin may cause damage to the immune systems, and 3'-dAdois also known to have various side effects. Thus, it would be verydesirable to have a radiosensitizing substance which is more stable,lower in toxicity, and more effective than 3'-dAdo.

On the other hand, in the field of chemotherapy of tumors, multipleanti-tumor agents have been combined to be used for the followingpurposes and effects:

(1) By using in combination a number of different agents selected fromthose of alkylating agents, antimetabolites, antibiotics and alkaloids,which show mutually no cross resistance and are different in mechanismof action, the anti-tumor effect can be enhanced additively orsynergistically against tumors which are composed of a mixture of tumorcells different in sensitivity to various agents.

(2) By using in combination anti-tumor agents different in the way theyattack tumor cells which are proliferating at random, various stages inthe cell cycle of tumor cells can be widely attacked to ensure completekilling of tumor cells.

(3) By using not only agents different in mechanism of action but alsothose having relatively similar mechanisms of action, a synergisticeffect can be expected. For example, by using in combination a number ofagents which are blocking a series of steps participating in DNAsynthesis, a strong synergistic effect can be exhibited.

(4) Each anti-tumor agent has its specific side effect. Thus, by usingin combination a number of agents with different side effects each in adosage less than the limit above which side effects appear, theanti-tumor effect can be expected to be increased additively orsynergistically while the side effects are dispersed.

By such a multi-agent combination treatment, it has been made possibleto obtain an effect which could not be produced by using a singleanti-tumor agent. However, each of the agents used in combination insuch an application is an anti-tumor agent which can be independentlyused.

There have also been various attempts to use in combination with ananti-tumor agent a compound which does not per se have an anti-tumoreffect for the purpose of strengthening the effect of the anti-tumoragent by preventing the anti-tumor agent from being inactivated inbodies. For example, it is known to use cytidine or uridine incombination with 1-β-D-arabinofuranosylcytosine (hereinafter referred toas "araC"), as disclosed in Japanese Laid-Open Patent Application No.24150/1980. It is also known to use tetrahydrouridine, which is aninhibitor against cytidinedeaminase, in combination with araC, asdisclosed in Cancer Research Vol.30, p. 2166-2172, 1970. Further, thereis known another method wherein 5-fluorouracil (hereinafter referred toas "5-FU") or a derivative thereof is combined with a pyrimidinecompound such as, for example, uracil, cytosine, thymine, orotic acid,5-bromouracil, 5-iodouracil, 1-acetyluracil,1-(2-tetrahydrofuryl)-uracil, 3-benzoyluracil,1-cyclohexycarbamoyluracil, 1-n-hexycarbamoyluracyl, uridine,2'-deoxyuridine, 5-bromo-2'-deoxyuridine, cytidine, or 2'-deoxycytidine.

With respect to a chemotherapeutics such as Bleomycin (hereinafterreferred to as "BLM") or 5-FU, PLDR is also recognized similarly as inthe case of radiotherapy, as reported in the Journal of the NationalCancer Institute, Vol.50, No.2, p.529-533, 1973.

SUMMARY OF THE INVENTION

In view of the above described state of the art, I have made extensivestudies with the aim of obtaining a radiosensitizing agent having aPLDR-inhibiting activity with low toxicity and good stability. As aresult, it has now been found that 3'-deoxyguanosine-5'-monophosphateand 3'-deoxyadenosine-5'-monophosphate, in addition to 3'-dAdo, haveexcellent radiosensitizing activities. I have also found that these3'-deoxynucleotides can also exhibit excellent effect in strengtheninganti-tumor effect in treatment of malignant tumors by chemotherapy.These findings have led to the development of the present invention.Thus, the present invention provides a method and a pharmaceutical agentto be used for strengthening the anti-tumor effect in treatment ofmalignant tumors by irradiation and/or an anti-tumor agent.

In one of its aspects, the present invention relates to a method forenhancement of anti-tumor effect, which comprises administering to atumor-bearing animal under the anti-tumor treatment an enhancer ofanti-tumor effect selected from the group consisting of3'-deoxyguanosine-5'-monophosphate, 3'-deoxyadenosine-5'-monophosphateand pharmaceutically-acceptable salts thereof.

The term "animal" as herein used means a human being or a lower animal.

The wording "under the anti-tumor treatment" means the state where atumor-bearing animal is being subjected to physical, chemical orphysicochemical treatment for suppressing tumors or where there isretained in that animal an influence due to such a treatment.Accordingly and more specifically, the enhancer is administered to theanimal before, simultaneously with, or after irradiation when theanti-tumor treatment comprises irradiation to a tumor site of theanimal. The enhancer is administered before, simultaneously with, orafter administration of an anti-tumor agent when the anti-tumortreatment comprises administration of an anti-tumor agent.

In another aspect of the present invention, it relates to a method fortherapy of tumors, which comprises administering to a tumor-bearinganimal under the anti-tumor treatment an enhancer of anti-tumor effectselected from the aforesaid group.

In still another aspect thereof, the present invention relates to anenhancer of anti-tumor effect, comprising an enhancer of anti-tumoreffect selected from the aforesaid group and a pharmaceuticallyacceptable carrier.

In a further aspect thereof, the present invention relates to achemotherapeutic composition for treating tumors, comprising ananti-tumor agent, an enhancer of anti-tumor effect selected from theaforesaid group, and a pharmaceutically acceptable carrier.

The enhancer is administered to a tumor-bearing animal under theanti-tumor treatment in accordance with the present invention and theanti-tumor effect due to the anti-tumor treatment is significantlyenhanced as evidenced by the data set forth in the followingexperiments.

DETAILED DESCRIPTION OF THE INVENTION Enhancers

The enhancer in accordance with the present invention is selected fromthe group consisting of 3'-deoxyguanosine-5'-monophosphate, hereinafterreferred to as 3'-dGuo-5'-MP, 3'-deoxyadenosine-5'-monophosphate,hereinafter referred to as 3'-dAdo-5'-MP, andpharmaceutically-acceptable salts thereof. Examples of thepharmaceutically-acceptable salts are their alkali metal (e.g., sodium,potassium, lithium) salts, alkaline earth metal (e.g., calcium,magnesium) salts and ammonium salts. These can of course be used incombination with each other or with other radiosensitizers or enhancersof chemotherapy.

Enhancement of anti-tumor effect

The pharmaceutical agent according to the present invention may be usedfor the purpose of enhancing the anti-tumor effect in the treatment of amalignant tumor, for which radiotherapy or chemotherapy by anti-tumoragents is to be applied, in combination with the treatments by thesetherapeutical methods.

In the case where the pharmaceutical agent of the present invention isused as a radiosensitizing agent for the purpose of enhancing the effectof radiotherapy, it may be administered before or after exposure, oreven during exposure, if the occasion permits it, to the irradiation inradiotherapy. As to radiotherapy per se, the use of specific method andconditions is not required, but conventional radiotherapy techniques maybe employed. By the use of the enhancer of the present invention incombination, it has become possible to apply radiotherapy withirradiation in the region of lower dosage than in the prior art. As theionizing radiations for radiotherapy, those generally employed such asX-rays, lineac high energy X-rays, betatron 32 MeV electron beams or.sup.α Coγ-rays may be used.

When used for the purpose of enhancing the anti-tumor effect inchemotherapy by an anti-tumor agent, the enhancer of the presentinvention may be administered simultaneously with, or after or beforeadministration of the anti-tumor agent. As anti-tumor agents whoseanti-tumor effect can be enhanced by the enhancer of the presentinvention, various kinds of agents, including those having activitysimilar to ionizing radiations as well as those having PLDR activity maybe mentioned. Examples of anti-metabolites include Methotrexate;6-mercaptopurine; 5-FU and its derivatives, such as, for example,5-fluorouridine, 5-fluoro-2'-deoxyuridine,1-β-D-arabinofuranosyl-5-fluorocytosine,1-(2-tetrahydrofuryl)-5-fluorouracil,1-(n-hexylcarbamoyl)-5-fluorouracil, 1-ethoxymethyl-5-fluorouracil,1-ethoxycarbonyl-5-fluorouracil, and b 5-fluoro-5'-deoxyuridine; andaraC and its derivatives, such as, for example, cyclocytidine, N⁴-palmitoyl araC, N⁴ -stearoyl araC, N⁴ -behenoyl araC,araC-5'-stearylphosphate, and araC-5'-oleylphosphate may be mentioned.Examples of anti-tumor antibiotics include BLM; Neocarzinostatin; andAnthracycline type antibiotics, such as, for example, Daunomycin,Adriamycin, and Aclacinomycin. Examples of alkylating agents includenitrosourea, such as, for example, ACNU, BCNU, CCNU, MCCNU;3'-[3-(2-chloroethyl)-3-nitrosoureido]-3'-deoxythymidine; and3'-(3-methyl-3-nitrosoureido)-3'-deoxythymidine. These anti-tumor agentsmay be administered by any method and in any dosage, which are notspecifically limited in combination with the enhancer of the presentinvention, but optimum conditions may suitably be selected for eachagent used.

The method for administration of the enhancer of the present inventionmay be either systemic administration or local administration. Variousdosages unit forms can be selected depending on the purposes of therapyand the methods of administration. For example, as the forms forsystemic administration, an oral administration form such as tablet,capsule, granule or solution, or a non-oral administration form such asinjection, suppository, etc., can be used. On the other hand, as a localadministration form, a slow-releasing-capsule, an ointment or aninjection can be used. In the preparing of such as dosage unit form, itis possible to make a preparation according to a conventional methodusing a pharmaceutically acceptable carrier. Various modifications inpreparation suitable for the object of the present invention may also beutilized. Further, as one embodiment of an enhancer of anti-tumor agentaccording to this invention, the anti-tumor agent to be used incombination and the active ingredient in the present pharmaceuticalagent may be formulated into a combined drug of the same dosage form.The compositions of the components in such a formulation may suitably bedetermined depending on the components employed.

As mentioned above, the enhancer of the present invention can beadministered simultaneously with administration of an anti-tumor agent.In such a case, instead of administering both chemicals separately, theycan be administered in the form of one formulation.

The enhancer in accordance with the present invention can be used whenthe animal is under the anti-tumor treatment comprising bothadministration of an anti-tumor agent and irradiation.

The enhancer of the present invention is used in an amount effective forenhancement of anti-tumor activity. More specifically, the dosage of thepharmaceutical agent of the present invention per day, which mayslightly differ depending on the active ingredient employed, in general,is desirably 20 to 3,000 mg for an oraladministration, 0.5 to 500 mg foran injection, and 20 to 2,000 mg for a suppository, as determined frombasic experiments on anti-tumor effectiveness. The optimum effectiveamount should be determined by judgement of a physician according to theirradiation used, its dosage, the anti-tumor agent used, its dosage, theconditions of disease, the affected part, etc.

The pharmacological effects of the pharmaceutical agents of the presentinvention are shown below with data from the tests of radiosensitizingeffect thereof.

EXPERIMENT 1 Radiosensitizing effect on the experimental tumors in mice

EMT-6 tumor cells (2×10⁵) were inoculated intradermally into the rightthighs of BALB/c-strain female mice (8 weeks old, 6 or more mice foreach group). When the tumor size reached 6 to 7 mm in diameter afterinoculation of the tumor cells, local irradiation with a 32 MeV electronbeam was carried out at 1,500 rad under no anesthesia and thereafter3'-dGuo-5'-MP and 3'-dAdo-5'-MP dissolved in physiological salinesolution, was administered intraperitoneally to each mouse in theindicated doses of respective agents. After these treatments, the tumorsizes were measured in tri-dimensional diameters every day or everyother day for 28 days, and compared with the control group with respectto the following items: ##EQU1##

The results are shown in Table 1 and Table 2, in which "cure" means thatthe tumors completely vanished during the observation period.

                                      TABLE 1                                     __________________________________________________________________________    Sensitizing effect of enhancer                                                on X-ray therapy of EMT-6 tumors                                                         Mean tumor                                                                    diameter at                                                                          2 weeks after treatment                                                                          3 weeks after treatment                             the time                                                                             Mean tumor         Mean tumor                                          of irradi-                                                                           diameter                                                                             Diameter                                                                            Volume                                                                              diameter                                                                             Diameter                                                                            Volume                      Treatment  ation (mm)                                                                           (mm)   ratio ratio (mm)   ratio ratio                       __________________________________________________________________________    Irradiation                                                                              6.80 ± 0.31                                                                       7.41 ± 0.51                                                                       1.09 ± 0.07                                                                      1.44 ± 0.23                                                                      0.29 ± 0.83                                                                       1.17 ± 0.13                                                                      2.06 ± 0.30              (1500 rad)                                                                    alone (n = 10)                                                                Irradiation +                                                                            6.46 ± 0.23                                                                       6.17 ± 0.90                                                                       0.93 ± 0.13                                                                      1.19 ± 0.32                                                                      5.95 ± 1.22                                                                       0.90 ± 0.18                                                                      1.45 ± 0.46              3'-dGuo-5'-MP                                                                 (100 mg/kg) (n = 9)                                                           Irradiation +                                                                            7.07 ± 0.23                                                                       4.75 ± 1.06                                                                       0.66 ± 0.14                                                                      0.63 ± 0.18                                                                      3.65 ± 1.24                                                                       0.49 ± 0.17                                                                      0.58 ± 0.27              3 '-dAdo-5'-MP                                                                (75 mg/kg) (n = 10)                                                           __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    Sensitizing effect of enhancer on                                             X-ray radiation on EMT-6 tumors                                               (on the 28th day after treatment)                                                            Less than 0.5                                                                         0.5 to less                                                                          1.0 to less                                                    of volume                                                                             than 1 of                                                                            than 2.0 of                                                                          2.0 or more                                             ratio exclud-                                                                         volume ratio                                                                         volume ratio                                                                         of volume                                               ing cure;                                                                             excluding                                                                            excluding                                                                            ratio exclud-                                           Tumor regres-                                                                         cure; Tumor                                                                          cure;  ing cure;                                               sion rate:                                                                            regression                                                                           regrowth:                                                                            regrowth:                                Treatment                                                                              Cure  rapid   rate: slow                                                                           slow   rapid                                    __________________________________________________________________________    Irradiation (1500                                                                      1/10 (10%)                                                                          0/10 (0%)                                                                             0/10 (0%)                                                                            3/10 (30%)                                                                           6/10 (60%)                               rad) alone                                                                    Irradiation +                                                                          2/9 (22%)                                                                           0/9 (0%)                                                                              2/9 (22%)                                                                            5/9 (56%)                                                                            0/9 (0%)                                 3'-dGuo-5'-MP                                                                 (100 mg/kg)                                                                   Irradiation +                                                                          5/10 (50%)                                                                          2/10 (20%)                                                                            0/10 (0%)                                                                            2/10 (20%)                                                                           1/10 (10%)                               3'-dAdo-5'-MP                                                                 (75 mg/kg)                                                                    __________________________________________________________________________

EXPERIMENT 2 Enhancement of the effect of anti-tumor agents on theexperimental tumors in mice

EMT-6 tumor cells (1×10⁶) were inoculated intradermally into the rightthighs of BALB/c-strain female mice (8 weeks old). On the 11th day afterinoculation of the tumor cells and thereafter, 3'-dGuo-5'-MP inphysiological saline solutions were administered intrapenitoneally tothe mice for 8 consecutive days in the dose of 50 mg/kg. Thepharmaceutical agents of the present invention were administered an hourafter administration of araC. After commencement of the treatment withpharmaceutical agents, the tumor sizes were measured and compared withthe control group.

The results are given in Table 3.

                                      TABLE 3                                     __________________________________________________________________________            Treatment with                                                                        Mean tumor diameter mm (ratio)                                Treatment                                                                             the enhancer                                                                          Immediately                                                                          7 days after                                                                          14 days after                                                                         21 days after                          with anti-                                                                            of the inven-                                                                         before commencement                                                                          commencement                                                                          commencement                           tumor agents                                                                          tion    treatment                                                                            of treatment                                                                          of treatment                                                                          of treatment                           __________________________________________________________________________    Control --      7.51 ± 0.53                                                                       10.07 ± 0.99                                                                       12.40 ± 1.36                                                                       16.18 ± 1.34                        (n = 9)                (1.34 ± 0.14)                                                                      (1.66 ± 0.21)                                                                      (2.16 ± 0.19)                       --      3'-duGuo-5'-MP                                                                        7.57 ± 0.64                                                                       9.83 ± 1.07                                                                        12.10 ± 2.00                                                                       15.40 ± 2.01                        (n = 10)                                                                              (50 mg/kg × 8)                                                                         (1.30 ± 0.13)                                                                      (1.61 ± 0.27)                                                                      (2.05 ± 0.32)                       araC    --      7.49 ± 0.63                                                                       9.63 ± 0.68                                                                        11.91 ± 1.21                                                                       15.49 ± 1.26                        (50 mg/kg × 8)   (1.29 ± 0.13)                                                                      (1.60 ± 0.20)                                                                      (2.08 ± 0.20)                       (n = 10)                                                                      araC    3'-dGuO-5'-MP                                                                         7.34 ± 0.62                                                                       8.28 ± 1.11                                                                        9.12 ± 3.67                                                                        12.07 ± 4.98                        (50 mg/kg × 8)                                                                  (50 mg/kg × 8)                                                                         (1.13 ± 0.15)                                                                      (1.26 ± 0.51)                                                                      (1.67 ± 0.68)                       (n = 9)                                                                       __________________________________________________________________________

EXPERIMENT 3 Acute toxicity test

The compounds of this invention were administered intraperitoneally toICR mice (male, 8 weeks old, 10 mice for each group), and the mice weresubjected to observation for a week.

The LD₅₀ value of 3'-dAdo-5'-MP was 500 mg/kg with confidence limitbeing 95% (417 to 600 mg/kg), and the LD₅₀ value of 3'-dGuo-5'-MP wasestimated to be more than 1000 mg/kg. On the other hand, the LD₅₀ (i.p.)of 3'-dAdo was 280 mg/kg with confidence limit being 95% (241.4 to 324.8mg/kg).

I claim:
 1. A method of enhancement of anti-tumor effect which comprisesadministering to a tumor-bearing animal under an anti-tumor treatment anenhancer of the anti-tumor effect selected from the group consisting of3'-deoxyguanosine-5'-monophosphate, 3'-deoxyadenosine-5'-monophosphateand pharmaceutically-acceptable salts thereof.
 2. The method ofenhancement of anti-tumor effect as claimed in claim 1 in which theanti-tumor treatment comprises irradiation of a tumor site of the animaland the enhancer is administered to the animal before, simultaneouslywith or after the irradiation.
 3. The method of enhancement ofanti-tumor effect as claimed in claim 2 in which the enhancer isadministered to the animal before the animal is subjected to theirradiation.
 4. The method of enhancement of anti-tumor effect asclaimed in claim 2 in which the enhancer is administered to the animalafter the animal has been subjected to the irradiation.
 5. The method ofenhancement of anti-tumor effect as claimed in claim 1 in which theanti-tumor treatment comprises administration to the animal of ananti-tumor agent to the animal and the enhancer is administered to theanimal before, simultaneously with or after the administration of theanti-tumor agent.
 6. The method of enhancement of anti-tumor effect asclaimed in claim 5 in which the anti-tumor agent is selected from thegroup consisting of anti-metabolites, anti-tumor antibiotics andalkylating agents.
 7. In a method of treating tumor in a tumor-bearinganimal, the improvement which comprises administering to a tumor-bearinganimal under the anti-tumor treatment an enhancer of anti-tumor effectselected from the group consisting of3'-deoxyguanosine-5'-monophosphate, 3'-deoxyadenosine-5'-monophosphateand pharmaceutically-acceptable salts thereof.
 8. The method of treatingtumor as claimed in claim 7 in which the anti-tumor treatment comprisesirradiation of a tumor site of the animal and the enhancer isadministered to the animal before, simultaneously with or after theirradiation.
 9. The method of treating tumor as claimed in claim 8 inwhich the enhancer is administered to the animal before the irradiation.10. The method of treating tumor as claimed in claim 8 in which theenhancer is administered after the irradiation.
 11. The method oftreating tumor as claimed in claim 7 in which the anti-tumor treatmentcomprises administration to the animal of an anti-tumor agent and theenhancer is administered before, simultaneously with or after theadministration of the anti-tumor agent.
 12. The method of treating tumoras claimed in claim 11 in which the anti-tumor agent is selected fromthe group consisting of anti-metabolites, anti-tumor antibiotics andalkylating agents.